The present invention relates to a method of detecting or quantitating endogenous wheat DNA in a test sample, and more particularly to a method of detecting or quantitating endogenous wheat DNA to be used when determining the contamination rate of genetically modified wheat contained in a food material or processed food.
In Japan more than 50 varieties of genetically modified crops (hereinafter, “genetically modified organism” or “GMO”) including maize, soybeans, and potatoes have undergone a safety assessment and have been approved for import and sale. Accordingly, a food product containing a GMO must be labeled based on the “Labeling Standard for Genetically Modified Foods issued by the Ministry of Agriculture, Forestry and Fisheries that was established in accordance with Article 7, paragraph 1 of the Quality Labeling Standard for Processed Foods and on Article 7, paragraph 1 of the Quality Labeling Standard for Fresh Foods” (Notification No. 517 of the Ministry of Agriculture, Forestry and Fisheries, 31 Mar. 2000), and on the “Enforcement of Ministerial Ordinance amending portions of Ministerial Ordinances on the Food Sanitation Law Enforcement Regulations and Concerning Compositional Standards, etc. for Milk and Milk Products” (Notice No. 79 of the Food Sanitation Department, Pharmaceutical and Medical Safety Bureau, Ministry of Health, Labor and Welfare 15 Mar. 2001).
In foreign countries, however, GMO crops may sometimes be cultivated together with non-GMO crops once the safety evaluation has been completed, and contamination may occur after harvest during the distribution process. The makers of food products and the like often subcontract the manufacturing of processed foods to manufacturing plants, and even though the contract may stipulate to use a non-GMO material, small amounts of a GMO may contaminate processed foods if a GMO is also used in the same plant. Therefore, to comply with the labeling obligations the makers of food products must inspect and analyze the finished processed food products to verify that they are not contaminated with a GMO.
Test methods for detecting a GMO in a test sample of a processed food, the raw material thereof, etc., include the detection of recombinant DNA by the polymerase chain reaction (hereinafter, “PCR”) and the detection of a recombinant protein by ELISA. In the case of processed foods, however, proteins often become denatured by heat and pressure, and they cannot be detected accurately by ELISA. Therefore, detection by PCR is commonly used.
Methods of laboratory analysis include the methods described in JAS Analytical Test Handbook, “Genetically Modified Food Test and Analysis Manual for Individual Products, Revised Second Edition (Non-Patent Document 1)” and those described in “Testing for Foods Produced by Recombinant DNA Techniques (Partially Revised)” (Notice No. 0618002 of the Food Sanitation Department, Ministry of Health, Labor and Welfare, Jun. 18, 2003). These documents describe that in the testing and analysis of a GMO it is necessary to perform PCR using a primer pair recognizing the endogenous DNA of each agricultural product and to verify that a PCR product of the predicted length is obtained for verification that DNA extracted from the test sample can be amplified by PCR. When quantitating a GMO contained in a test sample, a method is used of measuring the contamination rate of the modified crop based on the ratio of recombinant DNA to endogenous DNA that is always present in that crop.
In the case of maize for example, primer pairs have been developed that recognize each of the 5 strains of approved GMO varieties, along with a primer pair that recognizes the SSIIB gene region as an endogenous maize DNA (Non-patent document 1).
In “Testing for Foods Produced by Recombinant DNA Techniques (Partially Revised)” (Notice No. 1113001 of the Food Sanitation Department, Ministry of Health, Labor and Welfare, 13 Nov. 2003), during the process of performing quantitative PCR, the amplification products amplified by specific primer pairs targeting endogenous DNA and recombinant DNA of maize or soybean are ligated to a plasmid and used as a standard reference material. By performing PCR using this standard reference material, the ratio of the number of copies of recombinant DNA to the number of copies of endogenous DNA can be accurately determined in a test sample by fixed-time, quantitative PCR.
When there are a plurality of GMO strains, as in the case of maize, one particularly useful technique is to utilize a common standard reference material to measure the contamination rate of each strain, which can be done by using a standard reference material having endogenous DNA and DNAs specific to each strain incorporated into a single circular DNA molecule.
It is generally difficult to obtain genes specific to each strain, but once replicable DNA incorporating those genes has been prepared, it is possible to stably provide strain-specific DNA by replication thereof.
While no genetically modified products of common wheat have yet passed safety assessment, they are expected to appear on the market in the near future. Consequently, methods for detecting and quantitating endogenous wheat DNA and PCR primer pairs for use in such methods need to be developed to prepare for the distribution of GMO wheat.
The forms of genes found in wheat have diverse variations compared with other grains. That is because hexaploid, tetraploid, and diploid genotypes occur depending on the variety of wheat. For example, general common wheat is hexaploid (AA, BB, DD), and although each of the genes is similar, partial differences are found due to translocation and the like. On the other hand, durum wheat is tetraploid and does not contain genomic DD.
In terms of its genome structure and the nucleotide sequence of its encoded genes, wheat shares a high degree of homology with other cereal grains such as barley, rye and oats. These grains have a high level of homology with common wheat, and therefore the possibility of false detection is high.
Under these circumstances, it has been difficult in wheat to discover a DNA sequence satisfying the following four conditions: a) it is universally present in wheat varieties, b) the amount present (detected amount) will not be affected depending on the wheat variety, c) even if other grains are present, only wheat will be detected without cross-reactivity, and d) it will be amplified quantitatively by the PCR reaction.
WO 2005/097989 (Patent Document 1) discloses that a partial sequence of the Waxy gene (see Non-Patent Document 2), etc., can be used as endogenous wheat DNA satisfying such conditions.
Patent Document 1: WO 2005/097989
Non-Patent Document 1: JAS Analytical Test Handbook, “Genetically Modified Food Test and Analysis Manual for Individual Products, Revised Second Edition”
Non-Patent Document 2: Ainsworth C, et al., Plant Mol Biol. 1993 April; 22(1):67-82